The development of methods for detecting and sequencing nucleic acids is critical to the diagnosis of genetic, bacterial, and viral diseases. See Mansfield, E. S. et al. Molecular and Cellular Probes, 9, 145–156 (1995). DNA detection methods that employ gold nanoparticle probes, modified with oligonucleotides, to indicate the presence of a particular DNA are described in application number PCT/US00/17507, which is incorporated by reference herein in its entirety. Typically, oligonucleotides having sequences complementary to the nucleic acid to be detected are attached to a nanoparticle. The nanoparticle conjugate hybridized to the nucleic acid results in a detectable change resulting from the hybridization of the oligonucleotide on the nanoparticle to the nucleic acid target in solution.
In order to attach the oligonucleotide to the nanoparticle, the oligonucleotide, the nanoparticle or both, are functionalized. These methods are known in the art and include, for instance, the functionalization of oligonucleotides with alkanethiols at their 3′-termini or 5′-termini. Such functionalized nucleotides readily attach to gold nanoparticles.
A problem associated with nanoparticles derivatized with alkanethiol-oligonucleotides is that the oligonucleotides are easily detached from the nanoparticle surface when the system is heated above a certain temperature. Heating destabilizes and inactivates the nanoparticle-oligonucleotide probes. The oligonucleotides can also be displaced from the nanoparticle surface in the presence of other thiol containing compounds such as DTT.
There exists a need for oligonucleotide-nanoparticle probes, and bioconjugate-nanoparticle probes in general, that exhibit better anchoring of the oligonucleotide to the nanoparticle and are thus more stable and robust. Also needed are methods for preparing such complexes.